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Ensifer meliloti is a nitrogen-fixing symbiont of the alfalfa legume able to use heme as an iron source. The transport mechanism involved in heme acquisition in E. meliloti has been identified and characterized, but the fate of heme once inside the cell is not known. In silico analysis of E. meliloti 1021 genome revealed no canonical heme oxygenases although two genes encoding putative heme degrading enzymes, smc01518 and hmuS, were identified. SMc01518 is similar to HmuQ of Bradyrhizobium japonicum, which is weakly homologous to the Staphylococcus aureus IsdG heme-degrading monooxygenase, whereas HmuS is homolog to Pseudomonas aeruginosa PhuS, a protein reported as a heme chaperone and as a heme degrading enzyme. Recombinant HmuQ and HmuS were able to bind hemin with a 1:1 stoichiometry and displayed a Kd value of 5 and 4 µM, respectively. HmuS degrades heme in vitro to the biliverdin isomers IX-β and IX-δ in an equimolar ratio. The HmuQ recombinant protein degrades heme to biliverdin IX-δ only. Additionally, in this work we demonstrate that humS and hmuQ gene expression is regulated by iron and heme in a RirA dependent manner and that both proteins are involved in heme metabolism in E. meliloti in vivo.

Sinorhizobium meliloti has multiple systems for iron acquisition, including the use of haem as an iron source. Haem internalization involves the ShmR haem outer membrane receptor and the hmuTUV locus, which participates in haem transport across the cytoplasmic membrane. Previous studies have demonstrated that expression of the shmR gene is negatively regulated by iron through RirA. Here, we identify hmuP in a genetic screen for mutants that displayed aberrant control of shmR. The normal induction of shmR in response to iron limitation was lost in the hmuP mutant, showing that this gene positively affects shmR expression. Moreover, the HmuP protein is not part of the haemin transporter system. Analysis of gene expression and siderophore production indicates that disruption of hmuP does not affect other genes related to the iron-restriction response. Our results strongly indicate that the main function of HmuP is the transcriptional regulation of shmR. Sequence alignment of HmuP homologues and comparison with the NMR structure of Rhodopseudomonas palustris CGA009 HmuP protein revealed that certain amino acids localized within predicted beta-sheets are well conserved. Our data indicate that at least one of the beta-sheets is important for HmuP activity.

Herbaspirillum seropedicae Z67 is a nitrogen-fixing bacterium able to colonize the rhizosphere and the interior of several plants. As iron is a key element for nitrogen fixation, we examined the response of this microorganism to iron deficiency under nitrogen fixing conditions. We identified a H. seropedicae exbD gene that was induced in response to iron limitation and is involved in iron homeostasis. We found that an exbD mutant grown in iron-chelated medium is unable to fix nitrogen. Moreover, we provide evidence that expression of the nifH and nifA genes is iron dependent in a H. seropedicae genetic background.